I. Field of the Invention
The present invention relates generally to an RNA polymerase. More particularly, it provides a bacteriophage N4 virion RNA polymerase for synthesis of RNAs of desired sequences using single-stranded DNA templates.
II. Description of Related Art
The expression of a protein-encoding gene in a host cell involves transcription of messenger RNA (mRNA) from DNA by an RNA polymerase enzyme. Subsequently the mRNA is processed, involving recognition of a region of the 3′ UTR and addition of a tail of polyadenylate nucleotides to the 3′ end of the mRNA by polyadenylation enzymes. After transcription, the mRNA encounters ribosomes which associate with a region of the 5′ UTR of the mRNA and translocate in a 3′-ward direction along the mRNA. During translocation, amino acids are added to one another in sequence to form the polypeptide product of the protein-encoding gene. For prokaryotic transcription-translation, the Shine-Dalgarno sequence of the bacterial mRNA located about six to nine nucleotides before the initiation site for translation may be used for ribosome loading. This sequence is complementary to a sequence on the 3′ end of the 16S rRNA and stimulates ribosome binding to the mRNA. The base pairing between the Shine-Dalgarno sequence and the mRNA sequences serves to align the initiating AUG for decoding.
Transcription of DNA into mRNA is regulated by the promoter region of the DNA. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. RNA polymerases from different species typically recognize promoter regions comprised of different sequences. In order to express a protein-encoding gene in a host cell, either the promoter driving transcription of the protein-encoding gene must be recognized by a host RNA polymerase, or an RNA polymerase which recognizes the promoter driving transcription of the protein-encoding gene must be provided to the host cell (U.S. Pat. No. 6,218,145).
Most DNA-dependent RNA polymerases read double-stranded DNA, limiting RNA synthesis to systems in which a double-stranded DNA template is available. The synthesis of RNA using single-stranded DNA is not as common. Synthesizing RNA using a single-stranded DNA template immobilized on a solid support is described in U.S. Pat. No. 5,700,667.
Therefore, this invention provides an RNA polymerase that reads single-stranded DNA. Also provided is an RNA polymerase for which the promoter sequence is present upstream of the transcription initiation site and therefore is not transcribed by the polymerase.